Microencapsulation of magentic material using heat stabilization

ABSTRACT

Microencapsulation methods and products are provided. The method includes forming, at a first temperature, a emulsion which comprises aqueous microdroplets, including the agent (e.g., a magnetic material or drug) and a cross-linkable matrix material (e.g., a protein such as albumin), dispersed in a hydrophobic continuous phase comprising an oil and an oil-soluble surfactant, the first temperature being below the temperature effective to initiate cross-linking of the matrix material, and then heating the emulsion to a temperature and for a time effective to cause the matrix material to self-cross-link, to form microparticles comprising the agent encapsulated by the cross-linked matrix material.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Priority is claimed to U.S. Provisional Application No.60/415,493, filed Oct. 2, 2002. The application is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates generally to micro- and nano-encapsulationmethods, particularly for synthesizing magnetic microparticles, for usein biomedical or other applications.

[0003] Chemically cross-linked materials have been used in to formmicroparticles. The cross-linkable material forming the matrix can be asynthetic polymer or a natural polymer or protein, for example. Themicroparticles formed with these materials have been used for biomedicalapplications, primarily in the areas of drug delivery, immunoassay, andcell separation technologies. Chatterjee, et al., J. Mag. Magn. Mat.225:21 (2001) discloses a method of forming encapsulated particles bydissolving a polymer and a particular inorganic particle in an aqueoussolvent, forming an oil-in-water emulsion, and stabilizing the particlesusing chemical cross-linking. The stabilization by chemicalcross-linking can undesirably permit agglomeration. It therefore wouldbe desirable to avoid using a chemical cross-linker in a process forforming microparticles. It would be particularly desirable to make suchnanoparticles without requiring an emulsion polymerization reaction.

SUMMARY OF THE INVENTION

[0004] A method is provided for microencapsulating an agent. In oneaspect, the method comprises forming, at a first temperature, a emulsionwhich comprises aqueous microdroplets, including the agent and across-linkable matrix material, dispersed in a hydrophobic continuousphase comprising an oil and an oil-soluble surfactant, the firsttemperature being below the temperature effective to initiatecross-linking of the matrix material, and then heating the emulsion to atemperature and for a time effective to cause the matrix material toself-cross-link, to form microparticles comprising the agentencapsulated by the cross-linked matrix material. In one embodiment, theemulsion is formed by sonicating a mixture of an aqueous dispersion ofthe agent, in which the matrix material has been dissolved, with ahydrophobic liquid, such as an oil. In one embodiment, the step ofheating the emulsion comprises mixing the emulsion into a secondquantity of the hydrophobic liquid which has been heated. In oneembodiment, the method further includes isolating the microparticlesfrom the hydrophobic liquid.

[0005] In various embodiments, the cross-linkable matrix material isbiodegradable. In one embodiment, the matrix material comprises aprotein, such as an albumin, e.g., human serum albumin.

[0006] In various embodiments, the agent comprises a magnetic material.For example, the magnetic material could be one that includes iron,nickel, or cobalt. In one embodiment, the magnetic material comprisesmaghemite. In other embodiments, the agent comprises a drug, adiagnostic agent, an inorganic fertilizer, or an inorganic pigment.

[0007] In several embodiments, the agent comprises nanoparticles havinga number average diameter between 5 nm and 50 nm. The nanoparticles canbe superparamagnetic. For example, the nanoparticles could includenickel, cobalt, or iron (e.g., maghemite).

[0008] In one embodiment, the method produces microparticles having anumber average diameter between 100 and 1000 nm, e.g., between 300 and800 nm.

[0009] In one embodiment, thee hydrophobic continuous phase comprises anoil such as a vegetable oil (e.g., cottonseed oil) or a mineral oil(silicon oil).

[0010] In one embodiment, the oil soluble surfactant is selected fromthe group consisting of sorbitan esters, polyoxyethylene ethers,glycerol esters, sucrose esters, diblock copolymers of polyoxyethyleneand polyoxypropylene, and triblock copolymers of polyoxyethylene andpolyoxypropylene. In one embodiment, the oil soluble surfactantcomprises sorbitan sesquioleate.

[0011] In one embodiment, the method further comprises adsorbing aprotein-binding ligand onto the microparticles. Examples of such ligandsinclude avidin, biotin, streptavidin, and lectins. In one specificembodiment, the method further comprises modifying the microparticleswith lectin or other carbohydrate binding protein effective for couplingwith red blood cells.

[0012] In another aspect, a composition is provided which comprises amicroencapsulated agent made the methods described herein. For example,in one embodiment, the agent comprises maghemite in the form ofnanoparticles having a number average diameter between 5 nm and 50 nm,the matrix material comprises an albumin, and the microparticles have anumber average diameter between 300 and 800 nm.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 is a scanning electron micrograph of albumin magneticmicrospheres (taken in a dispersion) made by one embodiment of themicroencapsulation process described herein.

[0014]FIG. 2 is a scanning electron micrograph of albumin magneticmicrospheres (taken in powder form) made by one embodiment of themicroencapsulation process described herein.

[0015]FIG. 3 is a graph showing average particle size diameter versusthe number of particles of albumin magnetic microspheres, in adispersion, made by one embodiment of the microencapsulation processdescribed herein.

[0016]FIG. 4 is a graph showing average particle size diameter versusthe number of particles of albumin magnetic microspheres, in powderform, made by one embodiment of the microencapsulation process describedherein.

DESCRIPTION OF THE INVENTION

[0017] Improved microencapsulation methods have been developed formaking microparticles using a heat induced cross-linking process. In oneembodiment, the method includes forming, at a first temperature, aemulsion which comprises aqueous microdroplets, including the agent(e.g., a magnetic material or drug) and a cross-linkable matrix material(e.g., a protein such as albumin), dispersed in a hydrophobic continuousphase comprising an oil soluble surfactant, the first temperature beingbelow the temperature effective to initiate cross-linking of the matrixmaterial, and then heating the emulsion to a temperature and for a timeeffective to cause the matrix material to self-crosslink, to formmicroparticles comprising the agent encapsulated by the crosslinkedmatrix material. The heat treatment step was used to cause the formationof intermolecular bonds between adjacent matrix material chains, e.g.,the formation of disulfide bridges between the free SH groups onadjoining protein chains. Thus, the thermal denaturation is a curingprocess that yields a crosslinked polymer network structure.Microspheres comprised of heat-stabilized albumin encapsulatingmaghemite were found to be more stable and more polydisperse thanmicrospheres made by a chemical crosslinking process. Smaller moreuniform particles result from the present process, which is alsorelatively quicker and easier to use than conventional chemicalcrosslinking processes.

[0018] The microparticles made by the method generally are substantiallyspherical, i.e., microspheres. Exemplary ranges for the diameter of themicroparticles include from about 100 nm to about 3000 nm, morepreferably from about 100 nm to about 2000 nm. Exemplary ranges for thenumber average diameter of the microparticles are from about 300 nm toabout 800 nm. Filtration techniques can be used to isolate varioussubsets of sizes ranges of particles. The size can be critical to manyapplications. In particular, for in vivo applications, the size candetermine whether the particles accumulate and/or how the body removes(e.g., phagocytosis) or biodegrades them.

[0019] Stable magnetic microspheres can be made using the encapsulationprocess described herein. By “stable” is meant that the location of themagnetic particle should be predominately inside the microsphere. Incontrast, a microsphere consisting of magnetic particles adsorbed onto amicrosphere would not be considered stable, because the adsorbedparticle may detach from the surface during washing or a change intemperature or pH of the medium in which the microspheres operate.

The Emulsion

[0020] The first step(s) in the microencapsulation process includeforming a emulsion. The emulsion is a water-in-oil emulsion. Thedispersed phase, i.e., the microdroplets, includes the agent to beencapsulated and the crosslinkable matrix material in water or anaqueous solution. The microdroplets are dispersed in a continuous phasewhich includes a hydrophobic liquid and an oil soluble surfactant. Thecomponents of the mixture are combined and then emulsified usingtechniques and equipment known in the art. In one embodiment, theemulsification is done using conventional sonication equipment (e.g., anultrasonic homogenizer), while maintaining the emulsion at a temperaturebelow the temperature effective to initiate cross-linking of the matrixmaterial (e.g., in a processing vessel immersed in a cooling bath).

[0021] Ultrasonic mixing enables the formation of homogeneous emulsionwith very well dispersed phases. Preferably, the sonication is conductedat an amplitude between about 50% and about 60%. While higher amplitudesusually give smaller particles, such a process would generatesignificant amounts of undesirable heating, which could prematurelyinitiate crosslinking. Generally, sonication for about 30 seconds issufficient to achieve a homogenous mixture. As used herein,“sonication”, “ultrasonic mixing,” and “ultrasonication” all refer tothe technique known in the art that uses the application of acousticenergy to mix components together.

The Agent

[0022] The agent (i.e., the material to be encapsulated) can beessentially any microparticulate material that is stable across therange of temperatures encountered by the material in the presentencapsulation process, and that is substantially non-reactive with thematrix material and hydrophobic materials used. Examples of agentsinclude magnetic materials, drugs (i.e., therapeutic or prophylacticagents), diagnostic agents (e.g., contrast agents), inorganicfertilizers, or inorganic pigments.

[0023] In several embodiments, the agent comprises nanoparticles havinga number average diameter between 5 nm and 50 nm. The nanoparticles canbe superparamagnetic. For example, the nanoparticles could includenickel, cobalt, or iron (e.g., maghemite).

[0024] In one embodiment, the agent comprises or consists ofsuperparamagnetic nanoparticles. The superparamagnetic nanoparticlespreferably have an average diameter between about 5 nm and about 50 nm.The superparamagnetic nanoparticles can comprise iron, nickel, cobalt,and/or their alloys. One material for the superparamagneticnanoparticles is an iron oxide, such as magnetite, or more preferably,maghemite (λFe₂O₃). (Magnetite is susceptible to oxidation, whereasmaghemite is more stable to oxidation.) In other embodiments, themagnetic nanoparticles comprise an alloy or a mixture of elementalmaterials. For example, the magnetic nanoparticles can compriseiron-neodymium-boron.

[0025] The size of the agent particle preferably is between about 5 nmand about 100 nm. Particularly for magnetic particles, the smallersizes, e.g., between about 5 nm and about 10 nm, is preferred. A smallsize distribution is also preferred, as this can aid in determining thenecessary magnetic force to separate the nanoparticles from a fluidmedium.

[0026] The agent particles can be obtained using methods known in theart, depending, for example, on the particular agent to be encapsulated,and the desired size of the particles. Methods for making thesuperparamagnetic nanoparticles can be produced using any suitableprocess known in the art. For example, one technique for producing ironoxide nanoparticles involves co-precipitation and sonication. Onceobtained, the superparamagnetic nanoparticles or other agent particlescan be modified by treating them with an anionic surfactant to renderthem susceptible to microencapsulation, that is, to promote theircomplexation or attachment to the matrix material in the presentlydescribed encapsulation method.

[0027] The agent generally comprises between 5 and 40 wt % of themicroparticles. In one embodiment, the agent comprises between 25 and 35wt % of the microparticles. For example, the amount of magnetic materialmay depend on how much magnetic strength is desired for the finalencapsulated particles.

Cross-Linkable Matrix Material

[0028] The matrix material forms the bulk microparticle structure inwhich the agent is dispersed, encapsulated. It comprises a crosslinkablematerial that will crosslinks with itself upon heating. Preferably, thematrix material is biodegradable.

[0029] In one embodiment, the matrix material comprises a synthetic ornatural polymer. In another embodiment, the matrix material comprises aprotein. In one embodiment, the protein is an albumin. In a preferredembodiment, the matrix material comprises a human serum albumin.Examples of other matrix materials include bovine serum albumin, eggalbumin, and a variety of thermosetting polymers, including epoxies,polyurethanes, phenol/formaldehyde, and urea/formaldehyde resins.

Hydrophobic Liquid Phase

[0030] In one embodiment, the hydrophobic continuous phase comprises anoil which is substantially immiscible with the matrix material or theagent. The main criteria of the oil chosen is the flash point of theoil. The flash point of the oil should be much higher than thedenaturation temperature of the protein and for other hydrophobicsolvents, the boiling point should be much higher than the denaturationtemperature.

[0031] In one embodiment, the oil is biocompatible and non-toxic in thetrace amounts, if any, that may remain with the isolated microparticlesmade by the process described herein. The oil can be a vegetable oil ora mineral oil. Examples of suitable vegetable oils include cottonseedoil, rapeseed oil, and corn oil. Examples of suitable mineral oilsinclude silicon oil. In still other embodiments, the hydrophobic phaseis a mixture of toluene and chloroform, or isooctane and a mixture ofpetroleum ether with corn oil.

Oil Soluble Surfactant

[0032] The hydrophobic continuous phase further includes one or moresurfactants effective to reduce hydrophobicity of the matrix material tomitigate agglomeration of the microdroplets. Generally, the surfactantis mainly added in the oil phase in a water-in-oil emulsion to dispersethe microdroplets more evenly in the final emulsion, and the type andamount of surfactant determines whether nanospheres or microspheres areformed. Examples of suitable oil soluble surfactants include sorbitanesters (e.g., sorbitan trioleate, sorbitan monooleate, sorbitanmonolaurate, polyoxyethylene (20) sorbitan monolaurate, andpolyoxyethylene (20) sorbitan monooleate); polyoxyethylene ethers (e.g.,oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether,lauryl polyoxyethylene (4) ether); glycerol esters; sucrose esters; anddiblock and triblock copolymers of polyoxyethylene and polyoxypropylene(e.g., poloxamer 188 (Pluronic™ F-68), poloxamer 407 (Pluronic™ F-127),poloxamer 338). In one embodiment, the surfactant includes sorbitansesquioleate.

Heat-Stabilization

[0033] The second step(s) in the microencapsulation process include heatstabilization of the microdroplets. The heat stabilization step involvesheating the emulsion to a temperature and for a time effective to causethe matrix material to self-crosslink, to form microparticles comprisingthe agent encapsulated by the crosslinked matrix material. Preferably,this step also provides for the evaporation of water from themicrodroplets, which further rigidifies the microparticles.

[0034] The temperature and duration of heating is selected for optimumcrosslinking of the particular matrix material, without degrading theencapsulated agent. For example, a protein, such as albumin, can beheated to temperature of between 110 and 180° C. to effect crosslinking,over a period from 10 minutes to 75 minutes.

Further Processing

[0035] Optionally, a variety of protein-binding ligands can be adsorbedonto the microparticles. Representative examples of suitable ligandsinclude avidin, biotin, streptavidin, and lectins. For example, in oneembodiment, avidin-coating polymeric magnetic nanoparticles can be usedin the magnetic separation of red blood cells. The avidin can act as abridge that couples with polymeric nanoparticles modified withbiotinylated lectin. The lectin in the magnetic particle attaches to thesugar terminal on the red blood cell membrane, enabling the red bloodcell to be separated from its biological medium.

[0036] Optionally, the microparticles can be further encapsulated in apolymeric shell to provide additional or a different functionality. Forexample, it may be desirable to ensure that the magnetic material iswithin the particle and not on the surface of the particle. In addition,the polymeric coating may serve to functionalize the particle, forexample to couple it with a suitable ligand. For example, a polystyrenemagnetic particle can be functionalized with a carboxyl group orhydroxyl group by copolymerizing the first layer with acrylates orphenolics, in order to couple the particle with a protein.

Uses of the Microparticles

[0037] The microparticles made by the process described herein can beused in a variety of applications. Representative examples of suchapplications include magnetic separation processes, MRI, immunoassays,in vitro diagnostics, as a medium for transdermal drug delivery, andother biomedical processes, such as cell labeling, phagocytosis, sitespecific chemotherapy, radio immunoassay, affinity chromatography, andenzyme assay, and so forth. The particles could be useful in drugdelivery or diagnostic imaging (e.g., for the delivery of contrastagents). Other possible applications include calibration of flowcytometers, particle and hematology analyzers, confocal laser scanningmicroscopes, and zeta potential measuring instruments; supports forimmobilized enzymes, peptide synthesis, and separation phases forchromatography.

[0038] In a preferred embodiment, the magnetic nanoparticles are used ina batch or continuous process for the magnetic separation and isolationof blood components from a whole blood sample (e.g., blood cellseparation), for the treatment and clinical and laboratory testing ofsuch blood components. Such magnetic processes are described, forexample, in U.S. Pat. No. 6,129,848 to Chen, et al., which isincorporated herein by reference.

[0039] Biodegradable magnetic nanoparticles of heat stabilized albumin(e.g., made as described in Example 1 below) can be used to separate thered blood cells by using an external magnetic field. Certain types ofskin cancer treatment require complete separation of red blood cellsfrom the whole blood in order to get purely white blood cells which aretreated with a particular pharmaceutical (e.g., therapeutic) agent. Bymodification of these particles with avidin and biotinylated lectin,they can be attached the membrane of red blood cells, thus enablingthese coupled nanoparticles-cells to be separated by an externalmagnetic field. Similarly, the nanoparticles can be used to isolatewhite blood cells.

[0040] In another embodiment, the microparticles made as describedherein can be made to include one or more pharmaceutical agents, and theparticles can be utilized in the targeted delivery of drugs.

[0041] The invention can be further understood with the followingnon-limiting examples.

EXAMPLE 1 Synthesis of Albumin Magnetic Microspheres

[0042] Human serum albumin (HSA) magnetic microspheres containing 30%iron oxide particles were synthesized by a heat-stabilization process.HSA and cottonseed oil were obtained from Sigma Chemical Company. Ironoxide particles (maghemite with average size of 26 nm) were obtainedfrom Nanotechnology Corporation.

[0043] HSA (250 mg) was dissolved in a dispersion of 75 mg iron oxide in1 mL distilled water. The resulting solution/dispersion was added to 30mL cottonseed oil containing 0.2 mL sorbitan sesquioleate. The mixturewas shaken vigorously and then sonicated (using a Cole Parmer ultrasonichomogenizer) for three 30 s intervals at an amplitude of 60%. Thesonication process was performed at 4° C. using an ice-water bath. Theresulting (primary) emulsion was then added dropwise into 100 mLcottonseed oil heated at 130° C. and stirred at 1500 rpm, with theaddition completed in 10 minutes. The mixture was kept at 130° C. andstirred for another 15 minutes, to produce heat-stabilized microspheres.

[0044] The microspheres were then cooled and extracted with diethylether, and then washed by adding diethyl ether and centrifuging. Thedispersion of microspheres in ether was then filtered successively usingnylon filter membranes (Pall Specialty Chemicals) with pore sizes of 3,1.2, 0.8, 0.65, 0.45, and 0.30 μm. The retained microspheres werecollected, dried, and stored for subsequent testing.

EXAMPLE 2 Characteristics of Albumin Magnetic Microspheres

[0045] The microparticles made in Example 1 were examined using scanningelectron microscopy (SEM), atomic force microscopy (AFM), transmissionelectron microscopy (TEM), energy-dispersive X-ray analysis (EDXA),atomic absorption spectroscopy, and a superconducting quantuminterference device (SQUID) magnemometer.

[0046] SEM and AFM results show microspheres that are substantially notagglomerated and that have a textured (i.e., non-smooth,cauliflower-like) surface, which is believed to result from thecrosslinking process. See FIGS. 1 and 2.

[0047] In comparison to chemically stabilized microspheres made andtested in an earlier study, the heat stabilized microspheres made inExample 1 above possess a narrower size distribution. Histograms asobtained from SEM for the particle size distribution in dispersion andin powder form are shown in FIGS. 3 and 4, respectively.

[0048] The results of the EDXA showed that the magnetic particles wereessentially evenly distributed in the microsphere and did not cover theentire volume of the microsphere. This was supported by the TEM results.

[0049] Atomic absorption analysis showed approximately 32% iron in themicrospheres, which is significantly higher than that obtained inchemically stabilized microspheres made and tested in an earlier study.

[0050] The magnemometer analysis showed that microspheres havesuperparamagnetic characteristics.

[0051] Publications cited herein and the materials for which they arecited are specifically incorporated by reference. Modifications andvariations of the methods and devices described herein will be obviousto those skilled in the art from the foregoing detailed description.Such modifications and variations are intended to come within the scopeof the appended claims.

We claim:
 1. A method for microencapsulating an agent comprising:forming, at a first temperature, a emulsion which comprises aqueousmicrodroplets, including the agent and a crosslinkable matrix material,dispersed in a hydrophobic continuous phase comprising an oil and anoil-soluble surfactant, the first temperature being below thetemperature effective to initiate cross-linking of the matrix material;and heating the emulsion to a temperature and for a time effective tocause the matrix material to self-crosslink, to form microparticlescomprising the agent encapsulated by the crosslinked matrix material. 2.The method of claim 1, wherein the agent comprises a magnetic material.3. The method of claim 2, wherein the magnetic material comprises iron,nickel, or cobalt.
 4. The method of claim 3, wherein the magneticmaterial comprises maghemite.
 5. The method of claim 1, wherein theagent comprises nanoparticles having a number average diameter between 5nm and 50 nm.
 6. The method of claim 5, wherein the nanoparticles aresuperparamagnetic.
 7. The method of claim 6, wherein thesuperparamagnetic nanoparticles comprise iron, nickel, or cobalt.
 8. Themethod of claim 7, wherein the superparamagnetic nanoparticles comprisemaghemite.
 9. The method of claim 1, wherein the matrix material isbiodegradable.
 10. The method of claim 1, wherein the matrix materialcomprises a protein.
 11. The method of claim 1, wherein the matrixmaterial comprises an albumin.
 12. The method of claim 1, wherein thematrix material comprises a human serum albumin.
 13. The method of claim1, wherein the agent comprises a drug.
 14. The method of claim 1,wherein the agent comprises a diagnostic agent, an inorganic fertilizer,or an inorganic pigment.
 15. The method of claim 1, wherein themicroparticles have a number average diameter between 100 and 1000 nm.16. The method of claim 1, wherein the microparticles have a numberaverage diameter between 300 and 800 nm.
 17. The method of claim 1,wherein the oil is a vegetable oil or a mineral oil.
 18. The method ofclaim 1, wherein the oil soluble surfactant is selected from the groupconsisting of sorbitan esters, polyoxyethylene ethers, glycerol esters,sucrose esters, diblock copolymers of polyoxyethylene andpolyoxypropylene, and triblock copolymers of polyoxyethylene andpolyoxypropylene.
 19. The method of claim 1, wherein the oil solublesurfactant comprises sorbitan sesquioleate.
 20. The method of claim 1,wherein the emulsion is formed by sonication.
 21. The method of claim 1,wherein the step of heating the emulsion comprises mixing the emulsioninto a quantity of a heated oil.
 22. The method of claim 1, furthercomprising isolating the microparticles from the hydrophobic continuousphase.
 23. The method of claim 1, further comprising adsorbing aprotein-binding ligand onto the microparticles.
 24. The method of claim23, wherein the protein-binding ligand is selected from the groupconsisting of avidin, biotin, streptavidin, and lectins.
 25. A methodfor microencapsulating an agent comprising: forming, at a firsttemperature, a emulsion which comprises aqueous microdroplets, includingthe agent and a crosslinkable matrix material which comprises a protein,dispersed in a hydrophobic continuous phase comprising an oil solublesurfactant, the first temperature being below the temperature effectiveto initiate crosslinking of the protein; and heating the emulsion to atemperature and for a time effective to cause the protein toself-crosslink, to form microparticles comprising the agent encapsulatedby the crosslinked matrix material.
 26. The method of claim 25, whereinthe agent comprises maghemite.
 27. The method of claim 25, wherein theprotein comprises an albumin.
 28. The method of claim 23, furthercomprising modifying the microparticles with lectin or othercarbohydrate binding protein effective for coupling with red bloodcells.
 29. A composition comprising a microencapsulated agent made amethod comprising: forming, at a first temperature, a emulsion whichcomprises aqueous microdroplets, including the agent and a crosslinkablematrix material, dispersed in a hydrophobic continuous phase comprisingan oil soluble surfactant, the first temperature being below thetemperature effective to initiate crosslinking of the matrix material;and heating the emulsion to a temperature and for a time effective tocause the matrix material to self-crosslink, to form microparticlescomprising the agent encapsulated by the crosslinked matrix material.30. The composition of claim 29, wherein the agent comprises a magneticmaterial.
 31. The composition of claim 29, wherein the microparticleshave a number average diameter between 300 and 800 nm.
 32. Thecomposition of claim 29, wherein the agent is in the form ofnanoparticles having a number average diameter between 5 nm and 50 nm.33. The composition of claim 29, wherein the agent comprises a drug. 34.The composition of claim 29, wherein the matrix material comprises aprotein.
 35. The composition of claim 29, wherein the agent comprisesmaghemite in the form of nanoparticles having a number average diameterbetween 5 nm and 50 nm, the matrix material comprises an albumin, andthe microparticles have a number average diameter between 300 and 800nm.
 36. A composition comprising magnetic microparticles comprising:microparticles comprised of a crosslinked matrix material and anencapsulated magnetic material, wherein the microparticles have a numberaverage diameter between about 300 and about 800 nm.